This invention relates to compounds useful in the treatment of cancer. Particularly, this invention relates to anti-cancer drugs comprising an amino alcohol functionality, e.g. anthracyclines. More particularly, this invention relates to anthracycline aldehyde conjugates formed by reaction of an anthracycline with an aldehyde, e.g. formaldehyde.
Doxorubicin (adriamycin) continues to be one of the most important anti-cancer drugs available. It is a broad spectrum drug particularly useful in the treatment of Hodgkin""s disease, non-Hodgkin lymphomas, acute leukemias, sarcomas, and solid tumors of the breast, lung, and ovary (young, R. C. et al. (1981) New Engl. J. Med. 305:139-153). The closely related drug daunorubicin (daunomycin) is used primarily for the treatment of acute leukemia. A major problem associated with doxorubicin and daunorubicin chemotherapy is multi-drug resistance. Multi-drug resistance is characterized by resistance to several drugs developed by tumor cells upon treatment with one drug. Mechanisms proposed for tumor cell multi-drug resistance include overexpression of cell membrane proteins which enhance efflux of the drug, and overexpression of glutathione transferase which transforms xenobiotics to glutathione conjugates for excretion (Volm, M. (1991) Br. J. Cancer 64:700-704; Giai, M. et al. (1991) Eur. J. Gynaecol. Oncol. 12:359-73; Black, S. M. and Wolf (1991) Pharmac. Ther. 51:139-154; Serafino, A. et al. (1998) Anticancer Res. in press). Glutathione itself is also thought to be involved in resistance in a variety of tumors (Blair, S. L. (1997) Cancer Res. 57:152-155). Resistance to anthracycline anti-cancer antibiotics has been shown to involve a lower concentration of drug-produced reactive oxygen species, presumably resulting from overexpression of enzymes which destroy superoxide and hydrogen peroxide (Sinha, B. K. and Mimnaugh, E. G. (1990) Free Radicals Biol. Med.8:567-581.
In spite of intensive investigation of the mode of action of doxorubicin and daunorubicin, the events leading to cell death and differential cytotoxicity are not totally understood. This has hindered the development of new analogs which are both more effective and which overcome multi-drug resistance. Both drugs are excellent DNA intercalators, and have been shown to concentrate in the cell nucleus (Chaires, J. B. et al. (1996) Biochemistry 35:2047-2053; Egorin, M. J. et al. (1974) Cancer Res. 34:2243-2245; Coley, H. M. et al. (1993) Br. J. Cancer 67:1316-1323). Crystallographic data have established specific sequences as the sites of drug intercalation (Wang, A. H.-J. et al. (1987) Biochemistry 26:1152-1163; Frederick, C. A. et al. (1990) Biochemistry 29:2538-2549). The drugs are redox active through the quinone functionality and are substrates for one-electron redox enzymes such as xanthine oxidase, cytochrome P450 reductase, and mitochondrial NADH dehydrogenase (Pan, S. et al. (1981) Mol. Pharmacol. 19:184-186; Schreiber, J. et al. (1987) J. Am. Chem. Soc. 109:348-351; Schreiber, J. et al. (1987) J. Am. Chem. Soc. 109:348-351; Kappus, H. (1986) Biochem. Pharmacol. 35:1-6). Furthermore, reduction in the presence of molecular oxygen results in catalytic production of superoxide and hydrogen peroxide (Lown, W. J. et al. (1982) Biochem. Pharmacol. 31:575-581; Doroshow, J. H. (1983) Cancer Res.43:4543-4551; Sinha, B. K. (1989) Chem. Biol. Interact. 69:293-317). In an anaerobic environment, reduction leads to glycosidic cleavage to produce a quinone methide transient, long thought to be an alkylating agent for DNA (Kleyer, D. and Koch, T. H. (1984) J. Am. Chem. Soc. 106:2380-2387; Abdella, B. R. J. and Fisher, J. A. (1985) Envir. Health Perspect. 64:3-18; Gaudiano, G. et al. (1994) J. Am. Chem. Soc. 116:6537-6544; Moore, H. W. and Czerniak, R. (1981), Med. Res. Rev. 1:249-280). Currently, the most popular explanation for cytotoxicity is induction of topoisomerase-mediated DNA strand breaks through intercalation, with modulation through a signaling cascade involving a cell membrane receptor for doxorubicin (Liu, L. F. (1989) 58:351-375; Tritton, T. R. (1991) Pharmac. Ther. 49:293-301).
Recent reports from several laboratories have rekindled interest in the concept of drug alkylation of DNA via a redox pathway as an important cytotoxic event. Phillips and co-workers reported in a series of papers that in vitro reductive activation of doxorubicin and daunorubicin in the presence of DNA led to transcription blockages (Cullinane, C. R. (1994) Biochemistry 33:4632-8; Cullinane, C. (1994) Nucl. Acids Res. 22:2296-2303; van Rosmalen, A. (1995) Nucl. Acids Res. 23:42-50; Cutts, S. M. and Phillips, D. R. (1995) Nucl. Acids Res. 23:2450-6; Cutts, S. M. (1996)3J. Biol. Chem. 271:5422-9). These transcription blockages were attributed to the alkylation and crosslinking of DNA by reductively activated drug, possibly involving a quinone methide transient. The site of alkylation and crosslinking was proposed to be the 2-amino substituents of 2xe2x80x2-deoxyguanosines at the location. 5xe2x80x2-GpC-3xe2x80x2 in DNA. At about the same time, Skladanowski and Konopa established crosslinking of DNA by doxorubicin in HeLa S3 cells using a mild DNA denaturation-renaturation assay (Skladanowski, A. and Konopa, J. (1994) Biochem. Pharmacol. 47:2279-2287; Skladanowski, A. and Konopa, J. (1994) Biochem. Pharmacol. 47:2269-2278). They concluded that DNA crosslinks, although unstable to isolation, induced tumor cell apoptosis (Skladanowski, A. and Konopa, J. (1993) Biochem. Pharmacol. 46:375-382). We have recently demonstrated that the reported DNA alkylation and crosslinking does not involve the intermediacy of the quinone methide. The primary purpose of reductive activation of doxorubicin and daunorubicin is the production of superoxide and hydrogen peroxide (Taatjes, D. J. et al. (1996) J. Med. Chem. 39:4135-4138; Taatjes, D. J. et al. (1997) J. Med. Chem. 40, 1276-1286). These two reduced dioxygen species oxidize constituents in the medium to formaldehyde via Fenton chemistry (Taatjes, D. J. et al. (1997) Chem. Res. Toxicol. 10, 953-961). The resulting formaldehyde couples the 3xe2x80x2-amino group of intercalated doxorubicin or daunorubicin to the 2-amino group of deoxyguanosine via Schiff base chemistry. Thus, what Phillips and co-workers call a DNA xe2x80x9ccrosslinkxe2x80x9d by drug at 5xe2x80x2-GpC-3xe2x80x2, we describe as a xe2x80x9cvirtual crosslinkxe2x80x9d involving one covalent bond from formaldehyde and one intercalative-hydrogen bonding interaction with the opposing strand (Cullinane, C. R. (1994) Biochemistry 33:4632-8). This virtual crosslink is shown in Formula I for the DNA sequence 5xe2x80x2-CpGpC-3xe2x80x2 (Taatjes, D. J. et al. (1997) J. Med. Chem. 40, 1276-1286). 
There is a long-felt need in the art for improved anti-cancer drugs, particularly those with greater efficacy against resistant cancers. This invention provides such drugs.
This invention provides dimeric drug aldehyde conjugate compounds which are anti-cancer drugs, and pharmaceutically acceptable salts thereof, of Formula II: 
Formula B illustrates Formula A when Z1 is N(1) and Z2 is N(2). (The use of the numerals 1 and 2 inside parentheses is to distinguish one nitrogen atom from the other.) Z1, Z2, are the same or different heteroatoms, selected from the group consisting of N, S, P, Si, Se, and Ge. More preferably, Z1, Z2, are the same or different heteroatoms selected from the group consisting of N or S. Z1xe2x80x2 and Z2xe2x80x2, are the same or different heteroatoms selected from the group consisting of N, O, S, P, Si, Se, and Ge. Preferably, Z1xe2x80x2 and Z2xe2x80x2 are the same or different heteroatoms selected from the group consisting of N, S and O. Most preferably Z1 is N of an amino group and Z1xe2x80x2 is an N of an amino group or O of an alcohol group, and Z2 is N of an amino group and Z2xe2x80x2 is an N of an amino group or O of an alcohol group. If Z1xe2x80x2 is N, then preferably it is N of an amino group which is substituted with a non-hydrogen substituent, e.g., C1-20 alkyl or C1-20 acyl. If Z2xe2x80x2 is N, then preferably it is N of an amino group which is substituted with a non-hydrogen substituent.
Only the 1,2-dihetero substituted portion of the anti-cancer drug is shown in Formula II; the remainder of the drug is represented with are lines connected to a letter A or Axe2x80x2. A and Axe2x80x2 indicate that the remainder of the 1,2-dihetero-substituted portion of the anti-cancer drug may or may not be the same. In Formula II an example of A or Axe2x80x2 is the 7-deoxyaglycon portion of an anthtacycline attached at its 7-position to the remainder of the sugar.
Each R and Rxe2x80x3 is, independent of each other R and Rxe2x80x3, selected from the group consisting of xe2x80x94H, xe2x80x94OH, lower alkyl C1-6, lower alkenyl C1-6, C1-20 alkyl, C1-20 alkenyl, C1-20 acyl, aryl, hydroxylated alkyl, hydroxylated alkenyl, halogenated alkyl, halogenated alkenyl, silyl, sulfonyl, sulfonatoalkyl, alkylaryl, aralkyl, alkoxyalkyl, polyalkoxyalkyl, alkoxycarbonyl, carboxyalkyl, or aminocarbonyl.
Each n is 0 or 1, depending on the identity of Z1 and Z2xe2x80x2. Each p is 0, 1 or 2 depending on the identity of Z1xe2x80x2 and Z2xe2x80x2. One of skill in the art will understand that n and p are determined in part by the valence state of the heteroatom to which the substitutent is bonded. The valence state of the heteroatom is satisfied depending on the value of n and/or p. For example, of Z1xe2x80x2 is O (oxygen), then p is 0 (zero) as the valence state of oxygen calls for only two bonds to oxygen; hence, oxygen would not be substituted with an Rxe2x80x3.
Rxe2x80x2 is selected from the group consisting of xe2x80x94H, lower alkyl C1-6, lower alkenyl C1-6, C1-20 alkyl, C1-20 alkenyl, C1-20 acyl, aryl, hydroxylated alkyl, hydroxylated alkenyl, halogenated alkyl, halogenated alkenyl, silyl, sulfonyl, sulfonatoalkyl, alkylaryl, aralkyl, alkoxyalkyl, polyalkoxyalkyl, alkoxycarbonyl, carboxyalkyl, or aminocarbonyl.
All R, Rxe2x80x2, and Rxe2x80x3 can be optionally substituted, e.g. with halogens, hydroxyl groups, amines, amino groups, etc.
For all the formulas herein wherein there is more than one R, each R is selected independently of each other R. The same is true for Rxe2x80x2 and for Rxe2x80x3.
Those of ordinary skill in the art recognize can choose without undue experimentation acceptable and preferred R, Rxe2x80x2 and Rxe2x80x3 based on fundamental rules of organic chemistry. For example, if Z1xe2x80x2 is O (oxygen), then the Rxe2x80x3 bonded to Z1xe2x80x2 is preferably not xe2x80x94OH because this substitution would change the oxidation state of Z1xe2x80x2 and the resulting compound would be a hydroperoxide, which is unstable.
M1 and M2 are each a methylene, either or both of which can be substituted with xe2x80x94OH, lower alkyl C1-6, lower alkenyl C1-6, C1-20 alkyl, C1-20 alkenyl, C, 20 acyl, aryl, hydroxylated alkyl, hydroxylated alkenyl, halogenated alkyl, halogenated alkenyl, silyl, sulfonyl, sulfonatoalkyl, alkylaryl, aralkyl, alkoxyalkyl, polyalkoxyalkyl, alkoxycarbonyl, carboxyalkyl, or aminocarbonyl.
In Formula IIA, each of M1 and M2 is bonded to one of Z1 or Z2.
In Formula IIB each of M1 and M2 is bonded to one of N(1) or N(2).
Formula IIC illustrates that if M1 is bonded to Z1, then M2 is bonded to Z2. Formula IID illustrates that if M1 is bonded to Z2, then M2 is bonded to Z1.
More particularly, this invention provides dimeric drug aldehyde conjugates of the general structure shown in Formulas III A and B. 
The compounds of Formula III are preferably formed from drugs wherein the heteroatoms in the 1,2-Dihetero-substituted anti-cancer drug molecule are cis with respect to each other.
More particularly, this invention provides dimeric drug formaldehyde conjugates of the general structure shown in Formulas IV A and B. 
In Formula IV, A and Axe2x80x2 are as defined above. Formula IVB differs from Formula IVA in the stereochemistry of the bonds connecting the oxazolidine rings to the 6-membered glycosidic ring. Formulas IVA and B illustrate the two oxazolidine rings bound to each other via a methylene.
Also particularly provided are dimeric formaldehyde conjugates of Formulae VA, B, and C, wherein two drug cores are bound to each other via a diazadioxabicyclic ring. 
The compounds of Formula V are preferably formed from drugs wherein the heteroatoms in the 1,2-dihetero-substituted anti-cancer drug molecule are trans with respect to each other.
In Formula V, the regiochemistry of the heteroatoms can be changed, e.g., the O can be in the position of the N and vice versa. Furthermore, both heteroatoms can be N, such that each structure in Formula V would include 4 nitrogens. The difference between VA and VB is in the possible symmetry of the drug core of the 1,2-dihetero-substituted anti-cancer drug, represented by the A and Axe2x80x2.
Also particularly provided are formaldehyde conjugates of Formulas VIA, B, and C, wherein two drug cores are bound to each other via a diazadioxabicyclic ring. The letters A and Axe2x80x2 are as defined above and can be the same or different drug cores. In Formula VI an example of A and Axe2x80x2 is a 7-deoxyaglycon of an anthracycline attaced at its 7-position. Formulas VIA, B, and C differ among each other in the stereochemistry of the bonds attaching the diazadioxabicyclic ring to the 6-membered glycosidic rings. 
As in Formula V, the regiochemistry of the heteroatoms in Formula VI can be changed; e.g., the O can be in the position of the N and vice versa. Furthermore, three or all four heteroatoms can be N.
Formulas II through VI indicate that the dimeric drug aldehyde conjugates of this invention can comprise drug cores which are different from each other. For example, in Formula IV, A can be 7-deoxydaunorubicinon-7-yl and Axe2x80x2 can be 7-deoxydoxorubicinon-7-yl or A can be 7-deoxydoxorubicinon-7-yl and Axe2x80x2 can be 7-deoxydaunorubicinon-7-yl. Analogously, in Formula VI, A can be 7-deoxydaunorubicinon-7-yl and Axe2x80x2 can be 7-deoxydoxorubicinon-7-yl.
More particularly, this invention provides the compounds bis(3xe2x80x2-N-(3xe2x80x2-N,4xe2x80x2-O-methylenedoxorubicinyl))methane (the dimeric formaldehyde conjugate of doxorubicin) and bis(3xe2x80x2-N-(3xe2x80x2-N,4xe2x80x2-O-methylenedaunorubicinyl))methane (the dimeric formaldehyde conjugate of daunorubicin) which are dimeric oxazolidines, formaldehyde conjugates of the parent drugs, formed by reaction of formaldehyde with doxorubicin and daunorubicin, respectively. See Formula VII A and B, respectively. The dimeric formaldehyde conjugate of doxorubicin is much more cytotoxic to sensitive and resistant tumor cells than is the parent drug doxorubicin, and the dimeric formaldehyde conjugate of daunorubicin is much more cytotoxic to resistant tumor cells than is the parent drug daunorubicin. Also provided is the dimeric formaldehyde conjugate of epidoxorubicin, which consists of two molecules of epidoxorubicin bonded together with three methylene groups at the amino sugar in a 1,6-diaza-4,9-dioxabicyclo[4.4. ]undecane ring system. See Formula VII C. 
Drug aldehyde conjugates and particularly drug formaldehyde conjugates of anthracyclines besides doxorubicin, daunorubicin and epidoxorubicin are also disclosed. Methods for making drug aldehyde conjugates and drug formaldehyde conjugates are disclosed.
The aldehyde conjugates as described in Formulas II-VII show dimeric aldehyde conjugates, i.e., two 1,2-dihetero-substituted anti-cancer drug molecules are bonded by carbons derived from aldehydes. This invention also provides monomeric aldehyde conjugates, i.e., one 1,2-dihetero-substitued anti-cancer drug molecule is bonded by carbon derived from an aldehyde.
Monomeric drug aldehyde conjugates of 1,2-dihetero-substitued anti-cancer drugs of Formula VIII are provided: 
In Formula VIII R, Rxe2x80x3 and Rxe2x80x2 are as defined in Formula II. Each n is 0, 1 or 2. Each p is 0, 1 or 2. Z and Zxe2x80x2 are the same or different heteroatoms of an anti-cancer drug and are selected from the group consisting of N, S, O, P, Si, Se, and Ge. More preferably, Z and Zxe2x80x2 are, independently of each other, selected from the group consisting of N, S and O. Most preferably, one of Z and Zxe2x80x2 is N of an amino group and one of Z and Zxe2x80x2 is an O of an alcohol group. If both Z and Zxe2x80x2 are N, then preferably at least one of Z and Zxe2x80x2 is N of an amino group which is substituted with a non-hydrogen substituent, e.g., C1-20 alkyl or C1-20 acyl.
Monomeric drug aldehyde conjugates of Formula VIII are preferably formed by reaction of an aldehyde with a 1,2-dihetero substituted anti-cancer drug in which the 1,2-diheteroatoms are cis to each other.
1,2-dihetero substituted anti-cancer drugs in which the heteroatoms are trans to each other preferably undergo reaction to form a monomeric drug aldehyde conjugate of Formula IX. 
In Formula IX, R, Rxe2x80x2, Rxe2x80x3 are as defined above in Formula II. Z and Zxe2x80x2 are as defined above in Formula VIII. Each n is 0, 1 or 2. Each p is 0, 1, 2 or 3.
If the 1,2-dihetero substituted anti-cancer drug is an amino alcohol containing anti-cancer drug, and the heteroatoms are trans with respect to each other, then a monomeric drug aldehyde conjugate of Formula X is preferably formed. 
R and Rxe2x80x2 are as defined above.
This invention further provides pro-drugs which, after administration, release the monomeric aldehyde conjugates, e.g. the mono-oxazolidine or hydroxylmethylene compounds of this invention. The pro-drugs of 3xe2x80x2-N,4xe2x80x2-O-methylenedoxorubicin and 3xe2x80x2-N,4xe2x80x2-O-methylenedaunorubicin are more hydrolytically stable than the unfunctionalized/unprotected respective mono-oxazolidines as well as the dimeric formaldehyde conjugate of doxorubicin and the dimeric formaldehyde conjugate of daunorubicin, respectively. The pro-drugs of this invention include the mono-okazolidine compounds which have had functional groups added to them. These pro-drugs include but are not limited to compounds wherein the 3xe2x80x2-amino group of the mono-oxazolidine is functionalized/protected. This is accomplished, for example, by acylation or ethoxyformylation of the 3xe2x80x2-amino group. The resulting functional groups, e.g., amide and carbamate groups, are susceptible to hyrolysis in vivo, thereby releasing the mono-oxazolidine. Further, the functional groups can contain substituents which provide the compounds with desirable properties. For example, a carbamate functional group containing a t-butyl group or a hydrocarbon chain can be added to the drug to increase its lipophilicity, thereby facilitating incorporation of the drugs into a liposomal delivery system. Similarly, the hydroxylmethylene compounds (monomeric drug formaldehyde conjugates) of this invention, such as those formed by hydrolysis of the dimeric formaldehyde conjugate of epidoxorubicin, can be functionalized to form pro-drugs.
The compounds of this invention are useful in treating cancer. They are effective in inhibiting survival and/or growth of cancer cells and/or for inhibiting undesirable cell growth in general.
This invention further provides compositions and methods for clinical administration of aldehyde conjugates of this invention. In particular, compositions and methods of administering the dimeric formaldehyde conjugate of daunorubicin and the dimeric formaldehyde conjugate of doxorubicin in a liposomal delivery system are disclosed. A liposomal delivery system can be used to stabilize the formaldehyde conjugates of this invention, particularly the dimeric formaldehyde conjugate of doxorubicin, the dimeric formaldehyde conjugate of daunorubicin, and the respective mono-oxazolidines. Liposomes protect the drugs from undesirably premature hydrolysis of the formaldehyde derived conjugates and oxazolidine rings. Protection against premature hydrolysis is less of a concern with more hydrolytically stable conjugates, e.g. the dimeric formaldehyde conjugate of epidoxorubicin.
This invention further provides pharmaceutical and therapeutic compositions which contain a pharmaceutically or therapeutically effective amount of these conjugates and therapeutic methods and methods of treatment employing such methods. In particular, this invention relates to methods of treating cancer by administration of the anthracycline formaldehyde conjugates disclosed herein. A method of treatment of cancer when multidrug resistance has occurred by administration of the conjugates and compositions containing such conjugates is also provided.
This invention further provides for methods of treating cancer employing the compounds of this invention and pharmaceutical and therapeutic compositions and liposomal delivery systems.